Transcript Membrane Biophysics  Membrane  Protein rhodopsin The hydrophobic matching principle states that in the immediate vicinity of a peptide there is an accumulation of the.

Membrane Biophysics
 Membrane
 Protein
rhodopsin
The hydrophobic matching principle states
that in the immediate vicinity of a peptide
there is an accumulation of the lipid that is
hydrophobically best matched.
Outline
• Focus on model systems
– Experimental model systems
– Theoretical model systems
• Model systems do not have full biological
complexity
• Their value is in being able to do careful studies
to both discover and test conceptual ideas,
which hopefully are biologically relevant.
• Main topic: hydrophobic matching
Biological membranes:
different cellular
organelles have
different lipid and
protein membrane
compositions.
Why not study organelle membranes in their full complexity?
Cells determine the bilayer
characteristics of different
membranes by tightly controlling
their lipid composition.
We still have only sketchy information
on the lipid composition of organellar
membranes. In addition, we know
little about lipid–protein interactions at
the molecular level, let alone, lipid–
lipid interactions in complex mixtures.
Nat. Rev. Mol. Cell Biol. 2:504–513 (2001).
Protein – lipid interactions
• Focused on model
systems
• What is the simplest
possible model system?
• Answer: make the
bilayer membrane with
only one type of lipid,
and use a synthetic,
designed peptide.
J. Antoinette Killian.
Professor of
biophysical chemistry
of membranes at
Utrecht University
acetyl-GWWL(AL)nWWA-ethanolamide
n=3 -- 8
Hydrophobic matching principle
Hydrophobic
interactions play a
major role in stabilizing
membrane structures.
Transmembrane
peptides usually have
a hydrophobic region,
flanked by aromatic
and hydrophilic
residues.
Peptides are rigid
compared to lipids
FEBS Letters 458 (1999) 271-277
Hydrophobic matching principle
Exposure of protein hydrophobic residues
to water, or of lipid hydrophobic groups to
water, is unfavorable. What changes will
minimize the free energy?
1.
2.
1. Lipids adapt, forming a meniscus
2. Peptide is expelled from the membrane
Why might 2. be favored over 1.??
Answer: it costs energy to distort a membrane. How much energy?
Related to the bending modulus, compression modulus, etc. if we
think of the membrane as an elastic sheet (continuum description).
Slope gives area expansion modulus
Molecular dynamics simulations are used in
order to study the self-assembly process and
the physical properties of flexible membranes
composed of amphiphilic molecules. On
molecular scales, these membranes are
observed to be rather mobile and to have rough
surfaces arising from molecular protrusions,
i.e., from the relative displacements of
individual molecules. On length scales that are
only somewhat larger than the membrane
thickness, on the other hand, the membranes
are found to undergo smooth bending
undulations. In this way, our study provides the
first explicit connection between computer
simulations with molecular resolution and
elastic membrane models based on differential
geometry.
bending modulus
Phys. Rev. Lett. 82, 221, (1999).
Micropipette aspiration
Science 284,
1143 (1999).
(C and D) Microdeformation of a polymersome. The
arrow marks the tip of an aspirated projection as it is
pulled by negative pressure, DP, into the
micropipette. Aspiration acts to (i) increase membrane
tension, t = 1/2 DPRp/(1 - Rp/Rs), where Rp and Rs are the
respective radii of the micropipette and the outer spherical
contour; and (ii) expand the original, projected vesicle
surface area, Ao, by the increment DA.
Protein-Induced Bilayer
Deformations, and LipidInduced Protein Tilting
These are some of the
possibilities with a singlecomponent lipid bilayer. For
bilayers composed of more
than one type of lipid, there
are many more possibilities!
Some geometries of lipid phases
packed
spheres
Micellar
cubic
phase I1
packed
cylinders
hexagonal
phase H1
packed
planes
lamellar
phase La
cubic phases important for membrane
protein crystallization
blue, green, red regions are water
stacked bilayers
– why? Get
better signal to
noise in x-ray
diffraction
studies
TEM image of a
multilamellar vesicle onion
Caffrey, M. 2000. A lipid’s eye
view of membrane protein
crystallization in mesophases.
Curr. Opin. Struct. Biol.
10:486–497.
Caffrey, M. 2000. A lipid’s eye
view of membrane protein
crystallization in mesophases.
Curr. Opin. Struct. Biol.
10:486–497.
Electron density map constructed
from x-ray diffraction data.
Yang, L., and H. W.
Huang. 2002.
Observation of a
membrane fusion
intermediate structure.
Science. 297:1877–
1879.
More generally, inverted phases can be induced in
phospholipid membranes by dehydration, heating,
the addition of divalent cations, and the addition
of lipids of negative intrinsic curvature.
closely related to membrane fusion
Peptide-induced La to
inverted phase transition
B
A
S. O. Nielsen et. al., Biophys. J. 87, 2107 (2004) in
explanation of experimentally observed transition at 30:1
lipid:short-peptide concentration.
C
A. meniscus forms around peptides.
B. water fills the meniscus regions.
C. head groups rearrange to solvate
the water pores.
Structure of the inverted phase
6:1 lipid to peptide
concentration in HII phase.
Long peptides do not cause
this transition.
Biochemistry 35, 1037 (1996)
Biological context: membranes are
composed of many lipid species.
The hydrophobic matching principle states that in
the immediate vicinity of a peptide there is an
accumulation of the lipid that is hydrophobically
best matched.
Lipids come in
different shapes,
which determines
the self-assembled
structures they
prefer to form
(bilayers, micelles,
inverse micelles)
Why would a
biological membrane
contain non-bilayer
forming lipids?
Answer: for vesicle
budding and
membrane fusion
Nat. Rev. Mol. Cell Biol.
2:504–513 (2001).
Nat. Rev. Mol. Cell Biol.
2:504–513 (2001).
The organelles along the exocytic and endocytic transport routes are connected
by carrier vesicles that bud from one organelle and fuse with the next.
We will introduce the new and important
concept of protein/lipid sorting in membranes.
It is suggested that in any membrane, the hydrophobic mismatch
inherent to the protein and lipid composition may be released by a
process of protein aggregation or, more interestingly, via a general
mechanism of protein/lipid sorting.
This concept of hydrophobic mismatch-dependent protein/lipid sorting is
particularly attractive due to its inherent self- organizing character.
Nature Reviews in Molecular Cell Biology,
volume 2, page 504 (2001).
Sphingolipid domains sort proteins. A membrane that contains mostly
sphingomyelin, with or without cholesterol, is thicker than one
composed of phosphatidylcholine and cholesterol, which is in turn
thicker than a membrane of phosphatidylcholine alone. This implies
that sphingolipid–cholesterol domains are thicker than the
surrounding membrane. Cells probably use this feature to sort
membrane proteins that are destined for the plasma membrane from
Golgi proteins by the length of their transmembrane domains. For
example, the transmembrane domains of plasma membrane proteins
are 20 residues long, whereas those of Golgi proteins are only 15
residues long. Discrete increases in membrane thickness would allow
the sorting of various populations of membrane protein.
Hydrophobic mismatch induces
formation of a meniscus
S. O. Nielsen et. al., Biophys. J. 87, 2107 (2004)
S. O. Nielsen et. al., Biophys. J. 88, 3822 (2005)
• Maximal possible change in first shell
lipid length is small and represent only a
partial response to mismatch. (consistent
with experiments, slope < 1)
• Membrane thickens at intermediate
range
• The zero mismatch length can be
obtained and compared to results
obtained from free-energy calculations.
Free Energy Profile of Membrane Meniscus
against Hydrophobic Mismatch
1
DF (u0 )  k (u0  u0opt ) 2
2
Lipids do influence protein
function—the hydrophobic
matching hypothesis
revisited (Biochimica et
Biophysica Acta 2004, 1666,
205)
Xibing He, S.O. Nielsen, et. al.,
manuscript in preparation:
first time this effect has been
quantified by computer simulation
k = 650 kJ/mol
h0 1/ 2 1/ 4 3 / 4
( )  K L
2
Lipid tilting
J. Chem. Phys., 119, 7435 (2003)
Lipid tilting in the vertical direction
•
•
•
Shorter tubes make the lipids adopt a
more tilted and disorganized configuration
(compared to bulk).
Longer tubes make lipids acquire a
straighter configuration.
Straightening at intermediate distance due
to thickening of membrane.
Lipid tilting
Lipid tilting in the planar direction
•
•
For all peptide lengths the lipid’s head-totail vector points away from the peptide
(due to rigid peptide, consistent with
theory) with the shortest peptide giving the
biggest tilting.
Region of negative correlation due to void
resulting from positive meniscus (also
seen by B. Smit)
The polar-aromatic residues
Trp and Tyr have a specific
affinity for a region near the
lipid carbonyls.
Porin from the outer membrane of
Rhodobacter capsulatus (a) and the
membrane-bound form of the gramicidin A
dimer (b). Trp and Tyr residues are shown as
spacefilling models.
Trp
The membrane/water interfacial region is a
chemically complex environment and offers
opportunities for interactions with amino acid side
chains by dipole-dipole, charge, H-bonding, etc.
Tyr
What do 3-d structures say?
A central hydrophobic section is bordered on both
sides by aromatic belts, which were proposed to
interact favorably with the lipid headgroups.
Statistical analysis of porins and helix-bundle
membrane proteins such as cytochrome c
oxidase shows that Trp and Tyr (but not Phe)
are concentrated at the membrane/water
interface.
Charged residues are found only
outside the aromatic belts (aqueous
environment).
Phe
Trp
What chemical properties would be
responsible for a preferred localization of
aromatic and charged amino acid side
chains near the interfacial region?
Tyr
Must interact with a polar/apolar interface
Trp has a large fused aromatic ring which might
be buried in the hydrophobic part of the bilayer.
The amide group might localize in the more
polar environment at the interface. The amide
group can act as a hydrogen bond donor.
Phe
Tyr is smaller but has a similar ring system.
Phe is aromatic, but is completely hydrophobic
and is found in the transmembrane part of
membrane proteins.
Lys and Arg can snorkel
Arg
Lys
Biophysical approach which allows a direct comparison
between the interfacial interactions of different amino
acid side chains in transmembrane peptides:
WALP: GWW(LA)nLWWA
KALP: GKK(LA)nLKKA
These peptides are
incorporated into pure
lipid model membranes
(ie. one lipid type only)
Results suggest that Trp is
anchored rather rigidly to
the interface, whereas Lys
is much more flexible and
can be accommodated in a
larger range of bilayer
thicknesses than Trp.
Although biophysical studies have the obvious advantage that
detailed and fairly direct structural interpretations can often be
made, they are quite far removed from the complexities of a
biological membrane, and their relevance for understanding
what happens in vivo can be questioned.
However, the available methods for studying membrane proteins
in vivo rarely allow measurements to be made with the precision
required for locating individual amino acids relative to the
membrane–water interface.
There is one method that works in vivo:
you will be asked about this on your
take-home exam.
What is the biological relevance of these findings?
The strength of interfacial interactions might influence the
conformational flexibility of multi-spanning membrane proteins.
E. coli membrane channel protein TolC
Relatively rigid proteins that require no
or only very subtle structural changes
for functioning, such as gramicidin or
porins, might be conveniently and
stably anchored by Trp.
If functional properties require changes in
tilt or conformation, more flexible anchors,
at least on one side of the membrane,
might be more suitable.
This would be in agreement with the recent suggestion for single span
membrane proteins that Trp fulfills a stabilizing role as interfacial
anchoring residue, in particular at the trans-side of the membrane,
whereas Lys, as topological determinant, remains preferentially at the
cis-side (“positive-inside rule”), where it can act as a flexible anchor.